SUMMARY/ABSTRACT Adult skeletal muscle is endowed with significant regenerative capacity due to the presence of adult stem cells, called satellite cells (SCs). Very little is known about the developmental origin of these cells in humans and most of our knowledge derives from work performed in mouse. SCs are rare and they cannot be amplified in unlimited numbers, as they lose their regenerative potential in vitro. These issues have constituted a major roadblock for the study of their regenerative properties in humans and for the development of translational applications, such as cell therapy. The discovery that somatic cell reprogramming can be used to generate virtually endless numbers of cells (iPSCs) has brought new hope for developing strategies allowing to study these processes in human cells. We have recently developed efficient protocols to differentiate mouse and human embryonic pluripotent stem cells (ES and iPS) into striated muscle fibers and SCs in vitro [1, 6]. The present proposal aims to take advantage of these in vitro models combined with in vivo studies in mice to understand the development of the SC lineage in humans. In mouse, SCs constitute a heterogeneous population comprising a minor fraction of dormant cells with important regenerative capacity and a faster dividing larger population. Whether this heterogeneity is conserved in human is not known. Moreover, the developmental origin of this heterogeneity is not understood. Thus an important aim of the proposed project is to understand the developmental basis of this heterogeneity. We will compare the heterogeneity of mouse Pax7+ myogenic precursors developing in vivo and in vitro using a newly introduced method of single cell sequencing (InDrop) which allows to sequence thousands of cells in one experiment. A parallel analysis will be performed comparing human SCs differentiated in vitro to fetal and adult human muscle tissue. These studies are expected to reveal the developmental trajectories and the heterogeneity of the Pax7+ subpopulations in mouse and human SCs. We will also take advantage of our in vitro myogenic differentiation systems to perform high-resolution live- imaging studies of the development of the PAX7+ lineage. We will characterize poorly documented aspects of SC differentiation such as their entry in quiescence and their contribution to myotubes. Finally, we have identified Retinoic Acid (RA) and its co-activator complex WHHERE as a pathway able to induce reversible quiescence in PAX7+ cells differentiated in vitro. The role of RA in the differentiation and the maintenance of the PAX7+ lineage has not been investigated and we propose to characterize its function in vitro and in vivo in developing and mature SCs. We expect our work to shed light on the development and function of the human SC lineage, which could have significant implications for the development of cell-based therapies for muscle diseases.